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The cortactin gene (CTTN), encoding an actin-binding protein critically involved in cytoskeletal dynamics and endothelial cell (EC) barrier integrity, contains single nucleotide polymorphisms (SNPs) associated with severe asthma in Black patients. As loss of lung EC integrity is a major driver of mortality in the Acute Respiratory Distress Syndrome (ARDS), sepsis, and the acute chest syndrome (ACS), we speculated CTTN SNPs that alter EC barrier function will associate with clinical outcomes from these types of conditions in Black patients. In case-control studies, evaluation of a nonsynonymous CTTN coding SNP Ser484Asn (rs56162978, G/A) in a severe sepsis cohort (725 Black subjects) revealed significant association with increased risk of sepsis mortality. In a separate cohort of sickle cell disease (SCD) subjects with and without ACS (177 SCD Black subjects), significantly increased risk of ACS and increased ACS severity (need for mechanical ventilation) was observed in carriers of the A allele. Human lung EC expressing the cortactin S484N transgene exhibited: i) delayed EC barrier recovery following thrombin-induced permeability; ii) reduced levels of critical Tyr(486) cortactin phosphorylation; iii) inhibited binding to the cytoskeletal regulator, nmMLCK; and iv) attenuated EC barrier-promoting lamellipodia dynamics and biophysical responses. ARDS-challenged Cttn(+/−) heterozygous mice exhibited increased lung vascular permeability (compared to wild-type mice) which was significantly attenuated by IV delivery of liposomes encargoed with CTTN WT transgene but not by CTTN S484N transgene. In summary, these studies suggest that the CTTN S484N coding SNP contributes to severity of inflammatory injury in Black patients, potentially via delayed vascular barrier restoration.

We previously demonstrated involvement of NAMPT (nicotinamide phosphoribosyltransferase) in pulmonary arterial hypertension (PAH) and now examine NAMPT regulation and extracellular NAMPT’s (eNAMPT’s) role in PAH vascular remodeling. NAMPT transcription and protein expression in human lung endothelial cells were assessed in response to PAH-relevant stimuli (PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor], TGF-β1 [transforming growth factor-β1], and hypoxia). Endothelial-to-mesenchymal transition was detected by SNAI1 (snail family transcriptional repressor 1) and PECAM1 (platelet endothelial cell adhesion molecule 1) immunofluorescence. An eNAMPT-neutralizing polyclonal antibody was tested in a PAH model of monocrotaline challenge in rats. Plasma eNAMPT concentrations, significantly increased in patients with idiopathic pulmonary arterial hypertension, were highly correlated with indices of PAH severity. eNAMPT increased endothelial-to-mesenchymal transition, and each PAH stimulus significantly increased endothelial cell NAMPT promoter activity involving transcription factors STAT5 (signal transducer and activator of transcription 5), SOX18 (SRY-box transcription factor 18), and SOX17 (SRY-box transcription factor 17), a PAH candidate gene newly defined by genome-wide association study. The hypoxia-induced transcription factor HIF-2α (hypoxia-inducible factor-2α) also potently regulated NAMPT promoter activity, and HIF-2α binding sites were identified between −628 bp and −328 bp. The PHD2 (prolyl hydroxylase domain-containing protein 2) inhibitor FG-4592 significantly increased NAMPT promoter activity and protein expression in an HIF-2α–dependent manner. Finally, the eNAMPT-neutralizing polyclonal antibody significantly reduced monocrotaline-induced vascular remodeling, PAH hemodynamic alterations, and NF-κB activation. eNAMPT is a novel and attractive therapeutic target essential to PAH vascular remodeling.

Pharmacologic interventions to halt/reverse the vascular remodeling and right ventricular dysfunction in pulmonary arterial hypertension (PAH) remains an unmet need. We previously demonstrated extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a DAMP (damage-associated molecular pattern protein) contributing to PAH pathobiology via TLR4 ligation. We examined the role of endothelial cell (EC)-specific eNAMPT in experimental PH and an eNAMPT-neutralizing mAb as a therapeutic strategy to reverse established PH. Hemodynamic/echocardiographic measurements and tissue analyses were performed in Sprague Dawley rats exposed to 10% hypoxia/Sugen (three weeks) followed by return to normoxia and weekly intraperitoneal delivery of the eNAMPT mAb (1 mg/kg). WT C57BL/6J mice and conditional EC-cNAMPT ec−/− mice were exposed to 10% hypoxia (three weeks). Biochemical and RNA sequencing studies were performed on rat PH lung tissues and human PAH PBMCs. Hypoxia/Sugen-exposed rats exhibited multiple indices of severe PH (right ventricular systolic pressure, Fulton index), including severe vascular remodeling, compared to control rats. PH severity indices and plasma levels of eNAMPT, IL-6, and TNF- α were all significantly attenuated by eNAMPT mAb neutralization. Compared to hypoxia-exposed WT mice, cNAMPT ec−/− KO mice exhibited significantly reduced PH severity and evidence of EC to mesenchymal transition (EndMT). Finally, biochemical and RNAseq analyses revealed eNAMPT mAb-mediated rectification of dysregulated inflammatory signaling pathways (TLR/NF-κB, MAP kinase, Akt/mTOR) and EndMT in rat PH lung tissues and human PAH PBMCs. These studies underscore EC-derived eNAMPT as a key contributor to PAH pathobiology and support the eNAMPT/TLR4 inflammatory pathway as a highly druggable therapeutic target to reduce PH severity and reverse PAH.

Background There is a compelling unmet medical need for biomarker-based models to risk-stratify patients with acute respiratory distress syndrome. Effective stratification would optimize participant selection for clinical trial enrollment by focusing on those most likely to benefit from new interventions. Our objective was to develop a prognostic, biomarker-based model for predicting mortality in adult patients with acute respiratory distress syndrome. Methods This is a secondary analysis using a cohort of 252 mechanically ventilated subjects with the diagnosis of acute respiratory distress syndrome. Survival to day 7 with both day 0 (first day of presentation) and day 7 sample availability was required. Blood was collected for biomarker measurements at first presentation to the intensive care unit and on the seventh day. Biomarkers included cytokine-chemokines, dual-functioning cytozymes, and vascular injury markers. Logistic regression, latent class analysis, and classification and regression tree analysis were used to identify the plasma biomarkers most predictive of 28-day ARDS mortality. Results From eight biologically relevant biomarker candidates, six demonstrated an enhanced capacity to predict mortality at day 0. Latent-class analysis identified two biomarker-based phenotypes. Phenotype A exhibited significantly higher plasma levels of angiopoietin-2, macrophage migration inhibitory factor, interleukin-8, interleukin-1 receptor antagonist, interleukin-6, and extracellular nicotinamide phosphoribosyltransferase (eNAMPT) compared to phenotype B. Mortality at 28 days was significantly higher for phenotype A compared to phenotype B (32% vs 19%, p = 0.04). Conclusions An adult biomarker-based risk model reliably identifies ARDS subjects at risk of death within 28 days of hospitalization.

The lack of successful clinical trials in acute respiratory distress syndrome (ARDS) has highlighted the unmet need for biomarkers predicting ARDS mortality and for novel therapeutics to reduce ARDS mortality. We utilized a systems biology multi-“omics” approach to identify predictive biomarkers for ARDS mortality. Integrating analyses were designed to differentiate ARDS non-survivors and survivors (568 subjects, 27% overall 28-day mortality) using datasets derived from multiple ‘omics’ studies in a multi-institution ARDS cohort (54% European descent, 40% African descent). ‘Omics’ data was available for each subject and included genome-wide association studies (GWAS, n = 297), RNA sequencing (n = 93), DNA methylation data (n = 61), and selective proteomic network analysis (n = 240). Integration of available “omic” data identified a 9-gene set (TNPO1, NUP214, HDAC1, HNRNPA1, GATAD2A, FOSB, DDX17, PHF20, CREBBP) that differentiated ARDS survivors/non-survivors, results that were validated utilizing a longitudinal transcription dataset. Pathway analysis identified TP53-, HDAC1-, TGF-β-, and IL-6-signaling pathways to be associated with ARDS mortality. Predictive biomarker discovery identified transcription levels of the 9-gene set (AUC-0.83) and Day 7 angiopoietin 2 protein levels as potential candidate predictors of ARDS mortality (AUC-0.70). These results underscore the value of utilizing integrated “multi-omics” approaches in underpowered datasets from racially diverse ARDS subjects.

Therapeutic strategies to prevent or reduce the severity of radiation pneumonitis are a serious unmet need. We evaluated extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a damage-associated molecular pattern protein (DAMP) and Toll-Like Receptor 4 (TLR4) ligand, as a therapeutic target in murine radiation pneumonitis. Radiation-induced murine and human NAMPT expression was assessed in vitro, in tissues (IHC, biochemistry, imaging), and in plasma. Wild type C57Bl6 mice (WT) and Nampt(+/−) heterozygous mice were exposed to 20Gy whole thoracic lung irradiation (WTLI) with or without weekly IP injection of IgG(1) (control) or an eN-AMPT-neutralizing polyclonal (pAb) or monoclonal antibody (mAb). BAL protein/cells and H&E staining were used to generate a WTLI severity score. Differentially-expressed genes (DEGs)/pathways were identified by RNA sequencing and bioinformatic analyses. Radiation exposure increases in vitro NAMPT expression in lung epithelium (NAMPT promoter activity) and NAMPT lung tissue expression in WTLI-exposed mice. Nampt(+/−) mice and eNAMPT pAb/mAb-treated mice exhibited significant histologic attenuation of WTLI-mediated lung injury with reduced levels of BAL protein and cells, and plasma levels of eNAMPT, IL-6, and IL-1 β. Genomic and biochemical studies from WTLI-exposed lung tissues highlighted dysregulation of NFkB/cytokine and MAP kinase signaling pathways which were rectified by eNAMPT mAb treatment. The eNAMPT/TLR4 pathway is essentially involved in radiation pathobiology with eNAMPT neutralization an effective therapeutic strategy to reduce the severity of radiation pneumonitis.

Approximately 15%-20% of patients infected with SARS-CoV-2 coronavirus (COVID-19) progress beyond mild and self-limited disease to require supplemental oxygen for severe pneumonia; 5% of COVID-19-infected patients further develop acute respiratory distress syndrome (ARDS) and multiorgan failure. Despite mortality rates surpassing 40%, key insights into COVID-19-induced ARDS pathology have not been fully elucidated and multiple unmet needs remain. This review focuses on the unmet need for effective therapies that target unchecked innate immunity-driven inflammation which drives unchecked vascular permeability, multiorgan dysfunction and ARDS mortality. Additional unmet needs including the lack of insights into factors predicting pathogenic hyperinflammatory viral host responses, limited approaches to address the vast disease heterogeneity in ARDS, and the absence of clinically-useful ARDS biomarkers. We review unmet needs persisting in COVID-19-induced ARDS in the context of the potential role for damage-associated molecular pattern proteins in lung and systemic hyperinflammatory host responses to SARS-CoV-2 infection that ultimately drive multiorgan dysfunction and ARDS mortality. Insights into promising stratification-enhancing, biomarker-based strategies in COVID-19 and non-COVID ARDS may enable the design of successful clinical trials of promising therapies.

Recent innovations in translational research have ushered an exponential increase in the discovery of novel biomarkers, thereby elevating the hope for deeper insights into "personalized" medicine approaches to disease phenotyping and care. However, a critical gap exists between the fast pace of biomarker discovery and the successful translation to clinical use. This gap underscores the fundamental biomarker conundrum across various acute and chronic disorders: how does a biomarker address a specific unmet need? Additionally, the gap highlights the need to shift the paradigm from a focus on biomarker discovery to greater translational impact and the need for a more streamlined drug approval process. The unmet need for biomarkers in acute respiratory distress syndrome (ARDS) is for reliable and validated biomarkers that minimize heterogeneity and allow for stratification of subject selection for enrollment in clinical trials of tailored therapies. This unmet need is particularly highlighted by the ongoing SARS-CoV-2/COVID-19 pandemic. The unprecedented numbers of COVID-19-induced ARDS cases has strained health care systems across the world and exposed the need for biomarkers that would accelerate drug development and the successful phenotyping of COVID-19-infected patients at risk for development of ARDS and ARDS mortality. Accordingly, this review discusses the current state of ARDS biomarkers in the context of the drug development pipeline and highlight gaps between biomarker discovery and clinical implementation while proposing potential paths forward. We discuss potential ARDS biomarkers by category and by context of use, highlighting progress in the development continuum. We conclude by discussing challenges to successful translation of biomarker candidates to clinical impact and proposing possible novel strategies.

Rationale Despite the availability of multi-“omics” strategies, insights into the etiology and pathogenesis of sarcoidosis have been elusive. This is partly due to the lack of reliable preclinical models and a paucity of validated biomarkers. As granulomas are a key feature of sarcoidosis, we speculate that direct genomic interrogation of sarcoid tissues, may lead to identification of dysregulated gene pathways or biomarker signatures. Objective To facilitate the development sarcoidosis genomic biomarkers by gene expression profiling of sarcoidosis granulomas in lung and lymph node tissues (most commonly affected organs) and comparison to infectious granulomas (coccidiodomycosis and tuberculosis). Methods Transcriptomic profiles of immune-related gene from micro-dissected sarcoidosis granulomas within lung and mediastinal lymph node tissues and compared to infectious granulomas from paraffin-embedded blocks. Differentially-expressed genes (DEGs) were profiled, compared among the three granulomatous diseases and analyzed for functional enrichment pathways. Results Despite histologic similarities, DEGs and pathway enrichment markedly differed in sarcoidosis granulomas from lymph nodes and lung. Lymph nodes showed a clear immunological response, whereas a structural regenerative response was observed in lung. Sarcoidosis granuloma gene expression data corroborated previously reported genomic biomarkers (STAB1, HBEGF, and NOTCH4), excluded others and identified new genomic markers present in lung and lymph nodes, ADAMTS1, NPR1 and CXCL2. Comparisons between sarcoidosis and pathogen granulomas identified pathway divergences and commonalities at gene expression level. Conclusion These findings suggest the importance of tissue and disease-specificity evaluation when exploring sarcoidosis genomic markers. This relevant translational information in sarcoidosis and other two histopathological similar infections provides meaningful specific genomic-derived biomarkers for sarcoidosis diagnosis and prognosis.

Genetic factors are involved in acute respiratory distress syndrome (ARDS) susceptibility. Identification of novel candidate genes associated with increased risk and severity will improve our understanding of ARDS pathophysiology and enhance efforts to develop novel preventive and therapeutic approaches.To identify genetic susceptibility targets for ARDS.A genome-wide association study was performed on 232 African American patients with ARDS and 162 at-risk control subjects. The Identify Candidate Causal SNPs and Pathways platform was used to infer the association of known gene sets with the top prioritized intragenic SNPs. Preclinical validation of SELPLG (selectin P ligand gene) was performed using mouse models of LPS- and ventilator-induced lung injury. Exonic variation within SELPLG distinguishing patients with ARDS from sepsis control subjects was confirmed in an independent cohort.Pathway prioritization analysis identified a nonsynonymous coding SNP (rs2228315) within SELPLG, encoding P-selectin glycoprotein ligand 1, to be associated with increased susceptibility. In an independent cohort, two exonic SELPLG SNPs were significantly associated with ARDS susceptibility. Additional support for SELPLG as an ARDS candidate gene was derived from preclinical ARDS models where SELPLG gene expression in lung tissues was significantly increased in both ventilator-induced (twofold increase) and LPS-induced (5.7-fold increase) murine lung injury models compared with controls. Furthermore, SelplgThese findings identify SELPLG as a novel ARDS susceptibility gene among individuals of European and African descent.

Sarcoidosis is a granulomatous lung disorder of unknown cause. The majority of individuals with sarcoidosis spontaneously achieve full remission (uncomplicated sarcoidosis), however, ~20% of sarcoidosis-affected individuals experience progressive lung disease or cardiac and nervous system involvement (complicated sarcoidosis). We investigated peripheral blood mononuclear cell (PBMC) microRNA and protein-coding gene expression data from healthy controls and patients with uncomplicated or complicated sarcoidosis. We identified 46 microRNAs and 1,559 genes that were differentially expressed across a continuum of sarcoidosis severity (healthy control → uncomplicated sarcoidosis → complicated sarcoidosis). A total of 19 microRNA-mRNA regulatory pairs were identified within these deregulated microRNAs and mRNAs, which consisted of 17 unique protein-coding genes yielding a 17-gene signature. Pathway analysis of the 17-gene signature revealed Jak-STAT signaling pathway as the most significantly represented pathway. A severity score was assigned to each patient based on the expression of the 17-gene signature and a significant increasing trend in the severity score was observed from healthy control, to uncomplicated sarcoidosis, and finally to complicated sarcoidosis. In addition, this microRNA-regulated gene signature differentiates sarcoidosis patients from healthy controls in independent validation cohorts. Our study suggests that PBMC gene expression is useful in diagnosis of sarcoidosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology that poses significant challenges in early diagnosis and prediction of progression. Analyses of microRNA and gene expression in IPF have yielded potentially predictive information. However, the relationship between microRNA/gene expression and quantitative phenotypic value in IPF remains controversial, as is the added value of this approach to current molecular signatures in IPF. To identify biomarkers predictive of survival in IPF via a microRNA-driven strategy. We profiled microRNA and protein-coding gene expression in peripheral blood mononuclear cells from 70 IPF subjects in a discovery cohort. We linked the microRNA/gene expression level with the quantitative phenotypic variation in IPF, including diffusing capacity of the lung for carbon monoxide and the forced vital capacity percent predicted. In silico analyses of expression profiles and quantitative phenotypic data allowed the generation of two sets of IPF molecular signatures (unique for microRNAs and protein-coding genes) that predict IPF survival. Each signature performed well in a validation cohort comprised of IPF patients aggregated from distinct patient populations recruited from different sites. Resampling test suggests that the protein-coding gene based signature is comparable and potentially superior to published IPF prognostic gene signatures. In conclusion, these results highlight the utility of microRNA-driven peripheral blood molecular signatures as valuable and novel biomarkers associated to individuals at high survival risk and for potentially facilitating individualized therapies in this enigmatic disorder.

Significance: Acute respiratory distress syndrome (ARDS) is a severe, highly heterogeneous critical illness with staggering mortality that is influenced by environmental factors, such as mechanical ventilation, and genetic factors. Significant unmet needs in ARDS are addressing the paucity of validated predictive biomarkers for ARDS risk and susceptibility that hamper the conduct of successful clinical trials in ARDS and the complete absence of novel disease-modifying therapeutic strategies. Recent Advances: The current ARDS definition relies on clinical characteristics that fail to capture the diversity of disease pathology, severity, and mortality risk. We undertook a comprehensive survey of the available ARDS literature to identify genes and genetic variants (candidate gene and limited genome-wide association study approaches) implicated in susceptibility to developing ARDS in hopes of uncovering novel biomarkers for ARDS risk and mortality and potentially novel therapeutic targets in ARDS. We further attempted to address the well-known health disparities that exist in susceptibility to and mortality from ARDS. Critical Issues: Bioinformatic analyses identified 201 ARDS candidate genes with pathway analysis indicating a strong predominance in key evolutionarily conserved inflammatory pathways, including reactive oxygen species, innate immunity-related inflammation, and endothelial vascular signaling pathways. Future Directions: Future studies employing a system biology approach that combines clinical characteristics, genomics, transcriptomics, and proteomics may allow for a better definition of biologically relevant pathways and genotype–phenotype connections and result in improved strategies for the sub-phenotyping of diverse ARDS patients via molecular signatures. These efforts should facilitate the potential for successful clinical trials in ARDS and yield a better fundamental understanding of ARDS pathobiology.

IntroductionPseudogenes are paralogues of functional genes historically viewed as defunct due to either the lack of regulatory elements or the presence of frameshift mutations. Recent evidence, however, suggests that pseudogenes may regulate gene expression, although the functional role of pseudogenes remains largely unknown. We previously reported that MYLKP1, the pseudogene of MYLK that encodes myosin light chain kinase (MLCK), is highly expressed in lung and colon cancer cell lines and tissues but not in normal lung or colon. The MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. In this study, we further validate MYLKP1 as an oncogene via elucidation of the functional role of MYLKP1 genetic variants in colon cancer risk.MethodsProliferation and migration assays were performed in MYLKP1-transfected colon and lung cancer cell lines (H441, A549) and commercially-available normal lung and colon cells. Fourteen MYLKP1 SNPs (MAFs >0.01) residing within the 4 kb MYLKP1 promoter region, the core 1.4 kb of MYLKP1 gene, and a 4 kb enhancer region were selected and genotyped in a colorectal cancer cohort. MYLKP1 SNP influences on activity of MYLKP1 promoter (2kb) was assessed by dual luciferase reporter assay.ResultsCancer cell lines, H441 and A549, exhibited increased MYLKP1 expression, increased MYLKP1 luciferase promoter activity, increased proliferation and migration. Genotyping studies identified two MYLKP1 SNPs (rs12490683; rs12497343) that significantly increase risk of colon cancer in African Americans compared to African American controls. Rs12490683 and rs12497343 further increase MYLKP1 promoter activity compared to the wild type MYLKP1 promoter.ConclusionMYLKP1 is a cancer-promoting pseudogene whose genetic variants differentially enhance cancer risk in African American populations.

This article briefly reviews conventional biomarkers used clinically to (1) support a diagnosis and (2) monitor disease progression in patients with sarcoidosis. Potential new biomarkers identified by genome-wide screening and the approaches to discover these biomarkers are described.

Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH (n = 8), sarcoidosis patients without PH (n = 17), and healthy controls (n = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.

Acute respiratory distress syndrome (ARDS) is a major cause of morbidity and mortality in critically ill patients occurring in the context of sepsis and other systemic inflammatory disorders. Health disparities correlated with socioeconomic status and health care access disproportionately affect racial and ethnic minorities and lead to differences in prevalence and outcomes of ARDS. In addition, the role of genetic and environmental factors in critical illness outcomes has been examined. Multiple studies have highlighted differences in allelic variant frequencies between European descendant and African descent individuals. These allelic variants play an important role in inflammatory and infectious pathways in conditions that predispose to ARDS. In this chapter, we address race and ethnicity in the context of the complex genetic and environmental interactions that influence disparities in outcomes in ARDS in African American and Hispanic/Latino populations, groups most affected by health care disparities. We postulate that multiple factors contribute to poor outcomes in ethnic and racial minorities with ARDS and that precision medicine can be a realistic approach to overcome those disparities.

Background— We postulated that the hypoxic response in sickle cell disease (SCD) contributes to altered gene expression and pulmonary hypertension, a complication associated with early mortality. Methods and Results— To identify genes regulated by the hypoxic response and not other effects of chronic anemia, we compared expression variation in peripheral blood mononuclear cells from 13 subjects with SCD with hemoglobin SS genotype and 15 subjects with Chuvash polycythemia ( VHL R200W homozygotes with constitutive upregulation of hypoxia-inducible factors in the absence of anemia or hypoxia). At a 5% false discovery rate, 1040 genes exhibited >1.15-fold change in both conditions; 297 were upregulated and 743 downregulated including MAPK8 encoding a mitogen-activated protein kinase important for apoptosis, T-cell differentiation, and inflammatory responses. Association mapping with a focus on local regulatory polymorphisms in 61 patients with SCD identified expression quantitative trait loci for 103 of these hypoxia response genes. In a University of Illinois SCD cohort, the A allele of a MAPK8 expression quantitative trait locus, rs10857560, was associated with precapillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 mm Hg and pulmonary capillary wedge pressure ≤15 mm Hg at right heart catheterization (allele frequency, 0.66; odds ratio, 13.8; n=238). This association was confirmed in an independent Walk–Treatment of Pulmonary Hypertension and Sickle Cell Disease With Sildenafil Therapy cohort (allele frequency, 0.65; odds ratio, 11.3; n=519). The homozygous AA genotype of rs10857560 was associated with decreased MAPK8 expression and present in all 14 of the identified precapillary pulmonary hypertension cases among the combined 757 patients. Conclusions— Our study demonstrates a prominent hypoxic transcription component in SCD and a MAPK8 expression quantitative trait locus associated with precapillary pulmonary hypertension.

Sickle cell disease (SCD) is associated with pleiotropic clinical outcomes, the severity of which exhibits remarkable inter-individual variability. Investigations of the pathophysiology of SCD have focused on the adverse effects of vaso-occlusion, chronic inflammation, and hemolysis. SCD is also characterized by up-regulation of the hypoxic response to chronic anemia. We postulated that the up-regulated hypoxic response in SCD contributes to altered gene expression that might impact pulmonary hypertension, a complication associated with early mortality. To identify genes regulated by the hypoxic response and not other effects of chronic anemia, we compared expression variation in peripheral blood mononuclear cells from 13 sickle cell anemia untreated with hydroxyurea and 15 Chuvash polycythemia (CP) patients characterized by homozygous VHLR200W-induced constitutive up-regulation of hypoxia inducible factors in the absence of anemia or hypoxia. Gene expression of both cohorts was profiled on identical Affymetrix exon arrays. The degree and direction of differential gene expression were highly correlated between sickle cell anemia and CP (Spearman’s ρ = 0.73 between regression coefficients of differential gene expression), suggesting that 53% of expression variation in sickle cell anemia is related to hypoxic transcriptional responses. At 5% false discovery rate (FDR), 1040 genes exhibited a >1.15 fold change in both sickle cell anemia and VHLR200W homozygotes, among which 297 were up-regulated and 743 down-regulated. Hypoxia strongly induced inflammatory response pathways but suppressed T-cell activation in sickle cell anemia. MAPK8, encoding a mitogen-activated protein kinase important for stress-induced apoptosis, T-cell differentiation and inflammatory responses, was a hypoxia down-regulated gene and played a central role in hypoxic gene regulation in sickle cell anemia according to gene network analysis. To assess the genetic contribution to hypoxic transcriptional variation among SCD patients, we mapped expression quantitative trait loci (eQTL) for the 1,040 hypoxia response genes. Association mapping with a focus on local regulatory polymorphisms in 61 SCD patients identified eQTL for 103 of the hypoxia response genes at 5% FDR. We further tested the hypothesis that these hypoxic eQTL potentially underlie heterogeneity in risk of pulmonary hypertension in an additional SCD cohort (University of Illinois cohort). In this cohort, the A allele of an eQTL of MAPK8, rs10857560, was associated with pre-capillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 and pulmonary capillary wedge pressure ≤15 mm Hg at right heart catheterization (allele frequency=0.66; OR=4.4, P=0.00037, n=238). This association was confirmed in another independent cohort (Walk-PHaSST cohort) (allele frequency=0.65; OR=7.2, P=0.0025, n=519). The homozygous AA genotype of rs10857560, which was associated with decreased MAPK8 expression, was present in all 14 identified pre-capillary pulmonary hypertension cases among the combined 757 SCD patients (P = 6 x 10-6 by the Fisher exact test). Our study demonstrates a prominent hypoxic transcription component in SCD that in part contributes to pre-capillary pulmonary hypertension. Disclosures: No relevant conflicts of interest to declare.